Anti-fungal activity of a novel triazole, PC1244, against emerging azole-resistant Aspergillus fumigatus and other species of Aspergillus.

OBJECTIVES: The growing emergence of azole-resistant Aspergillus fumigatus strains worldwide is a major concern for current systemic antifungal treatment. Here we report antifungal activities of a novel inhaled triazole, PC1244, against a collection of multi-azole-resistant A. fumigatus strains. METHODS: MICs of PC1244 were determined for A. fumigatus carrying TR34/L98H (n = 81), TR46/Y121F/T289A (n = 24), M220 (n = 6), G54 (n = 11), TR53 (n = 1), TR463/Y121F/T289A (n = 2), G448S (n = 1), G432C (n = 1) and P216S (n = 1) resistance alleles originating from either India, the Netherlands or France. The effects of PC1244 were confirmed in an in vitro model of the human alveolus and in vivo in temporarily neutropenic, immunocompromised mice. RESULTS: PC1244 exhibited potent inhibition [geometric mean MIC (range), 1.0 mg/L (0.125 to >8 mg/L)] of growth of A. fumigatus strains carrying cyp51A gene mutations, showing much greater potency than voriconazole [15 mg/L (0.5 to >16 mg/L)], and an effect similar to those on other azole-susceptible Aspergillus spp. (Aspergillus flavus, Aspergillus terreus, Aspergillus tubingensis, Aspergillus nidulans, Aspergillus niger, Aspergillus nomius, Aspergillus tamarii) (0.18-1 mg/L). In TR34/L98H and TR46/Y121F/T289A A. fumigatus-infected in vitro human alveolus models, PC1244 achieved superior inhibition (IC50, 0.25 and 0.34 mg/L, respectively) compared with that of voriconazole (IC90, >3 mg/L and >10 mg/L, respectively). In vivo, once-daily intranasal administration of PC1244 (0.56-70 µg/mouse) to the A. fumigatus (AF91 with M220V)-infected mice reduced pulmonary fungal load and serum galactomannan more than intranasal posaconazole. CONCLUSIONS: PC1244 has the potential to become a novel topical treatment of azole-resistant pulmonary aspergillosis.

In Vitro and In Vivo Efficacy of a Novel and Long-Acting Fungicidal Azole, PC1244, on Aspergillus fumigatus Infection.

The antifungal effects of the novel triazole PC1244, designed for topical or inhaled administration, against Aspergillus fumigatus were tested in a range of in vitro and in vivo studies. PC1244 demonstrated potent antifungal activities against clinical A. fumigatus isolates (n = 96) with a MIC range of 0.016 to 0.25 µg/ml, whereas the MIC range for voriconazole was 0.25 to 0.5 µg/ml. PC1244 was a strong tight-binding inhibitor of recombinant A. fumigatus CYP51A and CYP51B (sterol 14α-demethylase) enzymes and strongly inhibited ergosterol synthesis in A. fumigatus with a 50% inhibitory concentration of 8 nM. PC1244 was effective against a broad spectrum of pathogenic fungi (MIC range, <0.0078 to 2 µg/ml), especially Aspergillus terreus, Trichophyton rubrum, Candida albicans, Candida glabrata, Candida krusei, Cryptococcus gattii, Cryptococcus neoformans, and Rhizopus oryzae PC1244 also proved to be quickly absorbed into both A. fumigatus hyphae and bronchial epithelial cells, producing persistent antifungal effects. In addition, PC1244 showed fungicidal activity (minimum fungicidal concentration, 2 µg/ml) which indicated that it was 8-fold more potent than voriconazole. In vivo, once-daily intranasal administration of PC1244 (3.2 to 80 µg/ml) to temporarily neutropenic, immunocompromised mice 24 h after inoculation with itraconazole-susceptible A. fumigatus substantially reduced the fungal load in the lung, the galactomannan concentration in serum, and circulating inflammatory cytokine levels. Furthermore, 7 days of extended prophylaxis with PC1244 showed in vivo effects superior to those of 1 day of prophylactic treatment, suggesting accumulation of the effects of PC1244. Thus, PC1244 has the potential to be a novel therapy for the treatment of A. fumigatus infection in the lungs of humans.

In Vivo Biomarker Analysis of the Effects of Intranasally Dosed PC945, a Novel Antifungal Triazole, on Aspergillus fumigatus Infection in Immunocompromised Mice.

PC945 is a novel triazole optimized for lung delivery, and the objective of this study is to determine the effects of intranasally dosed PC945 on Aspergillus fumigatus infection and associated biomarkers in immunocompromised mice. PC945, posaconazole, or voriconazole was administered intranasally once daily on days 0 to 3 (early intervention) or days 1 to 3 (late intervention) postinfection in temporarily neutropenic A/J mice infected intranasally with A. fumigatus, and bronchoalveolar lavage fluid (BALF) and serum were collected on day 3. The effects of extended prophylaxis treatment (daily from days -7 to +3 or days -7 to 0) were also compared with those of the shorter treatment regimens (days -1 to +3 or days -1 and 0). Early and late interventions with PC945 (2.8 to 350 µg/mouse; approximately 0.11 to ∼14 mg/kg of body weight) were found to inhibit lung fungal loads and to decrease the concentrations of galactomannan (GM) in both BALF and serum as well as several biomarkers in BALF (interferon gamma [IFN-γ], interleukin-17 [IL-17], and malondialdehyde) and serum (tumor necrosis factor alpha [TNF-α] and IL-6) in a dose-dependent manner and were >3- and >47-fold more potent than intranasally dosed posaconazole and voriconazole, respectively. Furthermore, extended prophylaxis with low-dose PC945 (0.56 µg/mouse; 0.022 mg/kg) was found to inhibit fungal loads and to decrease the concentrations biomarkers more potently than did the shorter treatment regimens. Thus, PC945 dosed intranasally once daily showed potent antifungal effects, and the effects of PC945 accumulated upon repeat dosing and were persistent. Therefore, PC945 has the potential to be a novel inhaled therapy for the treatment of A. fumigatus infection in humans.

In Vitro and In Vivo Antifungal Profile of a Novel and Long-Acting Inhaled Azole, PC945, on Aspergillus fumigatus Infection.

The profile of PC945, a novel triazole antifungal designed for administration via inhalation, was assessed in a range of in vitro and in vivo studies. PC945 was characterized as a potent, tightly binding inhibitor of Aspergillus fumigatus sterol 14α-demethylase (CYP51A and CYP51B) activity (50% inhibitory concentrations [IC50s], 0.23 µM and 0.22 µM, respectively) with characteristic type II azole binding spectra. Against 96 clinically isolated A. fumigatus strains, the MIC values of PC945 ranged from 0.032 to >8 µg/ml, while those of voriconazole ranged from 0.064 to 4 µg/ml. Spectrophotometric analysis of the effects of PC945 against itraconazole-susceptible and -resistant A. fumigatus growth yielded IC50 (determined based on optical density [OD]) values of 0.0012 to 0.034 µg/ml, whereas voriconazole (0.019 to >1 µg/ml) was less effective than PC945. PC945 was effective against a broad spectrum of pathogenic fungi (with MICs ranging from 0.0078 to 2 µg/ml), including Aspergillus terreus, Trichophyton rubrum, Candida albicans, Candida glabrata, Candida krusei, Cryptococcus gattii, Cryptococcus neoformans, and Rhizopus oryzae (1 or 2 isolates each). In addition, when A. fumigatus hyphae or human bronchial cells were treated with PC945 and then washed, PC945 was found to be absorbed quickly into both target and nontarget cells and to produce persistent antifungal effects. Among temporarily neutropenic immunocompromised mice infected with A. fumigatus intranasally, 50% of the animals survived until day 7 when treated intranasally with PC945 at 0.56 µg/mouse, while posaconazole showed similar effects (44%) at 14 µg/mouse. This profile affirms that topical treatment with PC945 should provide potent antifungal activity in the lung.

RV568, a narrow-spectrum kinase inhibitor with p38 MAPK-α and -γ selectivity, suppresses COPD inflammation.

Novel anti-inflammatory approaches targeting chronically activated kinase pathways in chronic obstructive pulmonary disease (COPD) are needed. We evaluated RV568, a p38 mitogen-activated protein kinase-α and -γ and SRC family kinase inhibitor, in cellular and in vivo models relevant to COPD and examined its safety and efficacy in COPD patients.The anti-inflammatory activities of RV568 were tested in primary cultured monocytes, macrophages and bronchial epithelial cells and in vivo in lipopolysaccharide and cigarette smoke-exposed murine models. RV568 was evaluated in a 14-day trial in COPD patients.RV568 showed potent anti-inflammatory effects in monocytes and macrophages, which were often greater than those of corticosteroids or the p38 inhibitor Birb796. RV568 combined with corticosteroid had anti-inflammatory effects suggestive of a synergistic interaction in poly I:C-stimulated BEAS-2B cells and in the cigarette smoke model. In COPD patients, inhaled RV568 (50 µg and 100 µg) improved pre-bronchodilator forced expiratory volume in 1 s (69 mL and 48 mL respectively) and significantly reduced sputum malondialdehyde (p<0.05) compared to placebo, although there were no changes in sputum cell counts. Adverse events during RV568 and placebo treatment were similar.RV568 shows potent anti-inflammatory effects on cell and animal models relevant to COPD. RV568 was well-tolerated and demonstrated a modest clinical benefit in a 14-day COPD clinical trial.

Effects of intranasally dosed posaconazole on fungal load and biomarkers in Aspergillus fumigatus infected immunocompromised mice.

Although anti-fungal triazoles are dosed orally or systemically for Aspergillus fumigatus infection, systemic adverse events and limited exposure of the lung cavity would make a topical treatment for the lung an attractive option. In this study, we examined the effects of intranasally dosed posaconazole on survival rates and biomarkers in A. fumigatus (itraconazole susceptible: ATCC13073 [Af]; or resistant: NCPF7100 [AfR]) infected, temporarily neutropenic A/J mice. Once daily treatment produced a dose-dependent improvement of survival of Af-infected mice (ED50 : 0.019 mg/mouse [approx. 0.755 mg/kg, in]), similar to its potency (ED50 : 0.775 mg/kg, po) after once daily oral dosing. For AfR infection, either intranasal or oral posaconazole was largely ineffective on survival, although the highest dose of intranasal treatment (0.35 mg/mouse) achieved 75% survival rate. Early intervention (treated on days 0, 1, 2 and 3 postinfection) and late intervention (treated on days 1, 2 and 3) with intranasal posaconazole (0.014-0.35 mg/mouse) demonstrated potent inhibition of lung fungal load and galactomannan levels in both bronchoalveolar lavage fluid (BALF) and serum as well as inflammatory cells, IFN-γ, IL-17 and malondialdehyde (MDA) in BALF. Thus, posaconazole when dosed intranasally once daily showed an improvement of survival equivalent to or better than oral treatment, and produced potent inhibition of fungal load and biomarkers.

[Anti-inflammatory Effects of a Src Inhibitor on the Murine Model of Asthma Exacerbation Induced by Ovalbumin and Lipopolysaccharide].

Asthma is often exacerbated by airway infection, and some patients with severe asthma may be unresponsive to conventional corticosteroid treatment. Src family kinases (SFKs) were recently implicated in the inflammatory responses of mice induced by allergen and bacterial toxin lipopolysaccharide (LPS). Therefore, we examined the effects of dasatinib (DAS), a Src inhibitor, on airway inflammation in mice induced by ovalbumin (OVA) and LPS. Male A/J mice were sensitized to OVA Day -14 and -7, challenged with intranasal OVA on Day 0, 2, 4, 6 and 8, and on Day 10, mice were also challenged with OVA via inhalation. Mice were treated intranasally with DAS or fluticasone propionate (FP), a glucocorticoid, twice daily for 3 d starting 1 d after OVA inhalation. Moreover, some mice were also administrated LPS 2 h after DAS or FP treatment to model of asthma exacerbation. One day after the last intervention, lung tissue and bronchoalveolar lavage fluid (BALF) were collected. DAS attenuated the accumulation of inflammatory cells and cytokines/chemokines in BALF induced by both OVA and OVA+LPS, while FP did not reduce accumulations induced by OVA+LPS. Therefore, targeting SFKs may be a superior therapeutic approach for asthma exacerbation by infection.

Dasatinib attenuates airway inflammation of asthma exacerbation in mice induced by house dust mites and dsRNA.

Asthma exacerbation is a significant clinical problem that causes resistance to corticosteroid therapy and elevated hospitalization risk. Src family kinases (SFKs) contribute to various steps of the immune response, such as airway inflammation in viral or bacterial infections and allergic asthma. Therefore, we determined the effects of dasatinib (DAS), a typical Src inhibitor, on a murine asthma exacerbation model induced by house dust mites (HDM) and synthetic analog of double-stranded RNA, poly(I:C). A/J mice were sensitized to intrapreneurial HDM twice every seven days and challenged with intranasal HDM once every second day for a total of six exposures, and/or exposed to poly(I:C) twice daily for three consecutive days. Drug treatments were performed twice daily for three days, starting one day after the last HDM challenge or 2 h before each poly(I:C) exposure. DAS improved poly(I:C)-induced acute inflammation dose-dependently. Both DAS and fluticasone propionate (FP) attenuated HDM-induced allergic airway inflammation. However, in HDM and poly(I:C) induced-asthma exacerbated mice, DAS significantly improved inflammatory cells in bronchoalveolar lavage fluid and histological changes in the lungs, whereas FP did not. Therefore, SFKs are important targets for controlling severe asthma refractory to conventional therapies.

Targeting phosphoinositide-3-kinase-delta with theophylline reverses corticosteroid insensitivity in chronic obstructive pulmonary disease.

RATIONALE: Patients with chronic obstructive pulmonary disease (COPD) show a poor response to corticosteroids. This has been linked to a reduction of histone deacetylase-2 as a result of oxidative stress and is reversed by theophylline. OBJECTIVES: To determine the role of phosphoinositide-3-kinase-delta (PI3K-δ) on the development of corticosteroid insensitivity in COPD and under oxidative stress, and as a target for theophylline. METHODS: Corticosteroid sensitivity was determined as the 50% inhibitory concentration of dexamethasone on tumor necrosis factor-α-induced interleukin-8 release in peripheral blood mononuclear cells from patients with COPD (n = 17) and compared with that of nonsmoking (n = 8) and smoking (n = 7) control subjects. The effect of theophylline and a selective PI3K-δ inhibitor (IC87114) on restoration of corticosteroid sensitivity was confirmed in cigarette smoke-exposed mice. MEASUREMENTS AND MAIN RESULTS: Peripheral blood mononuclear cells of COPD (50% inhibitory concentration of dexamethasone: 156.8 ± 32.6 nM) were less corticosteroid sensitive than those of nonsmoking (41.2 ± 10.5 nM; P = 0.018) and smoking control subjects (47.5 ± 19.6 nM; P = 0.031). Corticosteroid insensitivity and reduced histone deacetylase-2 activity after oxidative stress were reversed by a non-selective PI3K inhibitor (LY294002) and low concentrations of theophylline. Theophylline was a potent selective inhibitor of oxidant-activated PI3K-δ, which was up-regulated in peripheral lung tissue of patients with COPD. Furthermore, cells with knock-down of PI3K-δ failed to develop corticosteroid insensitivity with oxidative stress. Both theophylline and IC87114, combined with dexamethasone, inhibited corticosteroid-insensitive lung inflammation in cigarette-smoke-exposed mice in vivo. CONCLUSIONS: Inhibition of oxidative stress dependent PI3K-δ activation by a selective inhibitor or theophylline provides a novel approach to reversing corticosteroid insensitivity in COPD.

Relationship between anti-fungal effects and lung exposure of PC945, a novel inhaled antifungal agent, in Aspergillus fumigatus infected mice: Pulmonary PK-PD analysis of anti-fungal PC945.

PC945 is a novel antifungal agent, optimised for inhaled treatment. In this study, the relationship between antifungal effects of PC945 and its exposure in the lungs was investigated in Aspergillus fumigatus intranasally infected, temporarily neutropenic mice. Mice were given prophylactic PC945 intranasally once daily (0.56 µg/mouse) on either Day -7 to 0 (8 doses) or Day -1 to 0 (2 doses). Lung tissue, plasma and bronchoalveolar lavage (BAL) fluid were collected 24 or 72 h post A. fumigatus inoculation for biomarker and pharmacokinetic analyses. BAL cell pellets and supernatants were prepared separately by centrifugation. 8 prophylactic doses of PC945 were found to demonstrate significantly stronger antifungal effects (lung fungal burden and galactomannan (GM) in BAL and plasma) than prophylaxis with 2 doses. PC945 concentrations were below the limit of detection in plasma but readily measured in lung extracts. The concentrations were much higher after extended prophylaxis (709 and 312 ng/g of lung) than short prophylaxis (301 and 195 ng/g of lung) at 24 and 72 h post last dose, respectively, suggesting PC945 accumulation in whole lung after repeat dosing although it was likely to be a mixture of dissolved and undissolved PC945, meaning that the data should be interpreted with caution. Interestingly, low concentrations of PC945 were detected in BAL supernatant (6.6 and 1.9 ng/ml) whereas high levels of PC945 were measured in BAL cell pellets (626 and 406 ng/ml) at 24 and 72 h post last dose, respectively, in extended prophylaxis. In addition, the PC945 concentrations in BAL cells showed a statistically significant correlation with measured anti-fungal activities. These observations will be pursued, and it is intended that BAL cell concentrations of PC945 be measured in future clinical studies rather than standard measurement in BAL itself. Thus, PC945's profile makes it an attractive potential prophylactic agent for the prevention of pulmonary fungal infections.

Synthesis of water-soluble polyamine derivatives effective as N-methyl-D-aspartate receptor antagonists.

The novel water-soluble N-methyl-D-aspartate (NMDA) receptor antagonists, N-{4-[4-(4-Guanidinobutylamino)butylamino]butyl}-p-toluenesulfonamide trihydrochloride (1a, TsHSPMG), N-{4-[4-(4-Guanidinobutylamino)butylamino]butyl}butane-1-sulfonamide trihydrochloride (1b, BsHSPMG), N-{3-[4-(3-Guanidinopropylamino)butylamino]propyl}-p-toluenesulfonamide trihydrochroride (2a, TsSPMG) and N-{3-[4-(3-Guanidinopropylamino)butylamino]propyl}butane-1-sulfonamide trihydrochroride (2b, BsSPMG), were synthesized, and the effects of these polyamine derivatives on NMDA receptors were studied using voltage-clamp recordings of recombinant NMDA receptors expressed in Xenopus oocytes. Although spermine potentiates 153% and 310% of NMDA (NR1A/NR2B) receptors in the presence of saturated and unsaturated glycine, respectively, all the novel polyamine derivatives, TsHSPMG (1a), BsHSPMG (1b), TsSPMG (2a) and BsSPMG (2b), significantly inhibited NR1A/NR2B receptors in both conditions. The degree of NMDA receptor inhibition by TsHSPMG (1a) and BsHSPMG (1b) was stronger than that by TsSPMG (2a) and BsSPMG (2b).

[Inhibitory Effects of Dabigatran on Airway Inflammation Induced by Lipopolysaccharide in Mice].

Asthma and chronic obstructive pulmonary disease (COPD) are characterised by chronic inflammation in the lung that is associated with airway obstruction. Inhaled therapy with a combination of corticosteroid and a long-acting ß2-agonist is an effective anti-inflammatory medicine for asthma, but in patients with severe asthma and COPD fails to completely control these symptoms with current therapies. The inflammatory process in these diseases, which involves activation of the coagulation and fibrinolytic system in the lung, offers the opportunity for alternative anti-inflammatory therapies. In this study, we investigated the effects of anti-coagulants on lipopolysaccharide (LPS)-induced airway inflammation in mice. A/J mice were exposed to LPS, a bacterial endotoxin, intranasally and accumulation of inflammatory cells, TNF-α, C-X-C motif chemokine (CXCL) 1, and osteopontin in bronchoalveolar lavage fluid (BALF) was monitored by flow cytometry and an enzyme-linked immunosorbent assay. LPS exposure induced airway neutrophilia and accumulation of TNF-α, CXCL1, and osteopontin in BALF. This LPS-induced airway inflammation was not relieved using a corticosteroid, fluticasone propionate (FP), or a direct inhibitor of Factor Xa, rivaroxaban. In contrast, a direct thrombin inhibitor, dabigatran, inhibited LPS-induced airway neutrophilia and decreased inflammatory cytokine production in a dose dependent manner. Furthermore, combination of dabigatran and FP elicited stronger inhibition of LPS-induced airway inflammation. Therefore, these results suggest that dabigatran could be an effective new therapy for severe respiratory diseases.

Neuroprotection by tosyl-polyamine derivatives through the inhibition of ionotropic glutamate receptors.

Tosyl-polyamine derivatives such as N-{4-[4-(guanidinobutylamino)-butylamino]butyl}-4-methylbenzenesulfonamide trihydrochroride (TsHSPMG) have been found to strongly inhibit macroscopic currents through heteromeric N-methyl-D-aspartate (NMDA) receptors (NR1/NR2A, NR1/NR2B) and Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (homomeric glutamate receptor 1) receptors expressed in Xenopus laevis oocytes on voltage-clamp recording. In the present study, it was found that the inhibition of NMDA receptor activity induced by tosyl-polyamine derivatives was voltage-dependent. Some mutations located in the intracellular region of the channel pore, such as NR1 E621Q and NR2B W607L, reduced the inhibition by tosyl-polyamine derivatives, suggesting that tosyl-polyamine derivatives penetrate deeply into the channel pore of NMDA receptors. The neuroprotective effects of tosyl-polyamine derivatives against cell injury caused by NMDA were investigated in cultured rat hippocampal neurons. Addition of 1 microM TsHSPMG to medium ablated the neurotoxicity induced by NMDA, and a similar effect was observed with 30 microM memantine. The neuroprotective effects of tosyl-polyamine derivatives on NMDA-induced seizures in mice were also assayed. Intracerebroventricular or intravenous injection of TsHSPMG (0.1 or 0.5 mg/kg) decreased the seizures induced by intraperitoneal injection of NMDA in mice. These findings indicate that tosyl-polyamine derivatives exhibit neuroprotective effects not only in primary cultured neurons but also in mice.

[TNF-α Decreased Corticosteroid Responsiveness in Mice Models of Airway Inflammation Induced by Double Strand RNA and/or Tobacco Smoke Exposure].

Reduction of corticosteroid responsiveness is one of the important clinical problems in chronic obstructive pulmonary disease (COPD). In this study, we determined the effects of neutralization of tumor necrosis factor-α (TNF-α) on corticosteroid insensitivity in mice models of airway inflammation induced by poly(I:C) and tobacco smoke (TS) exposure. Mice (male A/J strain, 5 weeks old) were exposed to TS for 10 d, or TS for 11 d and poly(I:C) for 3 d. Anti-TNF-α antibody was intranasally treated once every other day 2 h before the TS exposure, and dexamethasone 21-phosphate (DEX) was treated 30 min before the TS or poly(I:C) exposure. On the next day of the last stimulation, mice were sacrificed. The combination treatment of DEX and TNF-α neutralization was significantly attenuated the increase of the numbers of inflammatory cells in BALF and the TNF-α mRNA expression levels induced by TS and poly(I:C) exposure, even though TNF-α neutralization alone had little effect. These data indicated that neutralization of TNF-α restores corticosteroid responsiveness. Therefore, our study suggests that targeting TNF-α signaling pathway provides a new therapeutic approach to corticosteroid refractory airway diseases.

Antifungal synergy of a topical triazole, PC945, with a systemic triazole against respiratory Aspergillus fumigatus infection.

Invasive pulmonary Aspergillosis is a leading cause of morbidity and mortality in immunosuppressed patients and treatment outcomes using oral antifungal triazoles remain suboptimal. Here we show that combining topical treatment using PC945, a novel inhaled triazole, with systemic treatment using known triazoles demonstrated synergistic antifungal effects against Aspergillus fumigatus (AF) in an in vitro human alveolus bilayer model and in the lungs of neutropenic immunocompromised mice. Combination treatment with apical PC945 and either basolateral posaconazole or voriconazole resulted in a synergistic interaction with potency improved over either compound as a monotherapy against both azole-susceptible and resistant AF invasion in vitro. Surprisingly there was little, or no synergistic interaction observed when apical and basolateral posaconazole or voriconazole were combined. In addition, repeated prophylactic treatment with PC945, but not posaconazole or voriconazole, showed superior effects to single prophylactic dose, suggesting tissue retention and/or accumulation of PC945. Furthermore, in mice infected with AF intranasally, 83% of animals treated with a combination of intranasal PC945 and oral posaconazole survived until day 7, while little protective effects were observed by either compound alone. Thus, the combination of a highly optimised topical triazole with oral triazoles potentially induces synergistic effects against AF infection.

[The Involvement of Src in Airway Inflammation Induced by Repeated Exposure to Lipopolysaccharide in Mice].

Corticosteroid insensitive airway inflammation is one of major barrier to effective managements of chronic airway diseases, such as chronic obstructive pulmonary disease (COPD) and severe asthma. The role of nonreceptor tyrosine kinase Src is important in airway inflammation in mice models of atopic asthma and COPD. Thus, in this study, we determined the effects of Src inhibitor, dasatinib, on airway inflammation induced by repeated intranasal exposure to lipopolysaccharide (LPS). Male mice (A/J strain, 5 weeks old) were intranasally exposed to LPS twice daily for 3 d, and dasatinib was intranasally treated 2 h prior to each LPS exposure. A day after the last stimulation, lungs and bronchoalveolar lavage fluid (BALF) were collected. Dasatinib attenuated the accumulation of inflammatory cells in lungs, and the increase in the numbers of inflammatory cells and the accumulation of cytokines/chemokines in BALF in a dose dependent manner. Therefore, this study suggested that targeting the Src can provide a new therapeutic approach for corticosteroid insensitive pulmonary diseases.

Differential effects of linear and cyclic polyamines on NMDA receptor activities.

The linear polyamine spermine enhances N-methyl-d-aspartate (NMDA) receptors activity at depolarized membrane potential and shows a voltage-dependent block. Spermine potentiates NMDA receptor currents in the presence of saturating concentrations of glutamate and glycine, but cyclic polyamines such as CP2323 do not. CP2323 inhibited the currents most potently amongst 10 kinds of cyclic polyamines tested. The inhibition was prominent at heteromeric NR1/NR2A and NR1/NR2B receptors but not at NR1/NR2C and NR1/NR2D receptors expressed in Xenopus oocytes. Inhibition by CP2323 was voltage-dependent, because the degree of inhibition was in the order -100mV>-70mV>-20mV. It was 10-100 times more prominent than inhibition by spermine. The inhibitory potency of both CP2323 and spermine was attenuated by the mutations around the vestibule of the channel pore at NR1 W563, N650, T807, and NR2B Y646. Inhibition by CP2323 was hardly affected by the mutations of NR1 N616 and E621, whereas inhibition by spermine was reduced by these mutations. The results suggest that CP2323 interacts with the vestibule region of the NMDA receptor and does not enter deep into the channel. Mutations of NR2B W607 greatly reduced the inhibition by CP2323 and spermine, suggesting that the mutation of this residue may cause the change of the channel structure. Neuroprotective effects of cyclic polyamines against cell damage caused by NMDA were compared with those of spermine in cultured rat hippocampal neurons. Addition of CP2323, but not spermine, into the medium attenuated the neurotoxicity induced by NMDA. These results indicate that CP2323 functions as a channel blocker of the NMDA receptor.

N1-Nonyl-1,4-diaminobutane ameliorates brain infarction size in photochemically induced thrombosis model mice.

Inhibitors for polyamine oxidizing enzymes, spermine oxidase (SMOX) and N1-acetylpolyamine oxidase (PAOX), were designed and evaluated for their effectiveness in a photochemically induced thrombosis (PIT) mouse model. N1-Nonyl-1,4-diaminobutane (C9-4) and N1-tridecyl-1,4-diaminobutane (C13-4) competitively inhibited the activity of PAOX and SMOX in a manner comparable to N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL72527), an irreversible inhibitor of both enzymes. The two compounds were then tested for their effects in the PIT model. Both intraperitoneal (i.p.) and intracerebroventricular (i.c.v.) administration of C9-4 decreased infarct volumes significantly. By contrast, C13-4 reduced the volume of brain infarction by i.c.v. administration, but no reduction was observed after i.p. administration. C9-4 administered by i.p. injection reduced the volume of brain infarction significantly at doses of more than 3 mg/kg, and the dosage of 5 mg/kg or 10 mg/kg demonstrated the most potent effect and were more effective than equivalent doses of the other inhibitors such as MDL72527 and N-benzylhydroxylamine. I.P. injection of 5 mg/kg of C9-4 provided a therapeutic time window of longer than 12 h. This report demonstrates that C9-4 is a potent inhibitor of the polyamine oxidizing enzymes and is useful lead compound for candidate drugs with a long therapeutic time window, to be used in the treatment of ischemic stroke.

Cleft-type cyclophanes confer neuroprotection against excitatory neurotoxicity in vitro and in vivo through inhibition of NMDA receptors.

The cleft-type cyclophanes (ACCn, DNCn and TsDCn) were found to strongly inhibit macroscopic currents at heteromeric NMDA receptors (NR1/NR2) but not AMPA receptors expressed in Xenopus oocytes at voltage-clamp recording. The inhibition by cleft-type cyclophanes was voltage-dependent, because the inhibition was larger at -100 mV than at -20 mV. Mutations at NR1 N650, located in the vestibule of the channel pore, reduced the inhibition by DNCn and TsDCn, suggesting that the residue (N650) interacts with these cleft-type cyclophanes. Cell toxicity of TsDCn on SH-SY5Y cells was slightly weaker than that of memantine. The neuroprotective effects of cleft-type cyclophanes against cell damage caused by NMDA were investigated in cultured rat hippocampal neurons. Addition of 10 microM DNCn or TsDCn into the medium ablated the neurotoxicity induced by NMDA, and a similar effect was also observed with memantine. The neuroprotective effects of cleft-type cyclophanes were then assayed on NMDA-induced seizures in mice. Intracerebroventricular injection of TsDCn (5 mg/mouse) decreased the seizure induced by intraperitoneal injection of NMDA (115 mg/kg) in mice. The results demonstrate that these cleft-type cyclophanes interact directly with the extracellular mouth of the NMDA channel pore and exhibit neuroprotective effects on NMDA-induced excitatory toxicity in primary cultured neurons and mice.

Cyclophane and acyclic cyclophane: novel channel blockers of N-methyl-D-aspartate receptor.

The effects of cyclophanes (CPCn, CPPy and TGDMAP) and acyclic cyclophane (ATGDMAP) on various glutamate receptors were studied with these receptors expressed in Xenopus oocytes using voltage-clamp recording. CPCn, CPPy, TGDMAP and ATGDMAP were found to inhibit macroscopic currents at heteromeric NMDA receptors (NR1/NR2), but not Ca(2+)-permeable AMPA receptors (GluR1), Ca(2+)-nonpermeable AMPA receptors (GluR1/GluR2) and metabotropic glutamate receptors (mGluR1alpha). The inhibition of NR1/NR2A receptors by these compounds was more potent than those of the other NMDA receptor subtypes. At a resting potential (-70 mV), the IC(50) values of CPCn, CPPy, TGDMAP and ATGDMAP for NR1/NR2A receptors were 0.5+/-0.1, 1.0+/-0.2, 8.0+/-0.8 and 4.9+/-0.5 microM, respectively. The inhibition by these compounds was voltage-dependent, that is, the degree of inhibition was in the order of negative holding potentials, -100 mV>-70 mV>-20 mV. Results of experiments using mutant NR1 and NR2 subunits identified residues that influence block by CPCn. The inhibition by CPCn was not altered significantly in the mutants at the critical asparagines in the M2 loop, NR1 N616, NR2B N615 and NR2B N616, these residues are known to form the narrowest region of the channel and the binding site of Mg(2+). However, mutations at NR1 N650, located in the vestibule of channel pore, and NR1 D669, located in the extracellular region, reduced the inhibition by CPCn, suggesting that these amino acid residues interact with CPCn. These results suggest that CPCn interacts directly with the mouth or vestibule of the ion channel, like a lid.